Name: coherent anti-stokes raman spectroscopy (cars) application for imaging myelination in brain slices
Uploaded: 2022-07-22T14:00:00.000+00:00
Duration: 4 min 8 s
Description: Watch this Scientific Journal Video about Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices at JoVE.com

Supported by NIH R01 DC 17924, R01 DC 18401 (Klug), and NIH 1R15HD105231-01, T32DC012280, and FRAXA (McCullagh). The CARS imaging was performed in the Advanced Light Microscopy Core part of the NeuroTechnology Center at the University of Colorado Anschutz Medical Campus supported in part by NIH P30 NS048154 and NIH P30 DK116073.

All experiments complied with all applicable laws, National Institutes of Health guidelines, and were approved by the University of Colorado Anschutz Institutional Animal Care and Use Committee.
1. Animals
<ol>
	<li>Use C57BL/6J (stock #000664) mice (Mus musculus) obtained from The Jackson Laboratory or Mongolian gerbils (Meriones unguiculatus) originally obtained from Charles River.</li>
</ol>
2. Tissue preparation
<ol>
	<li>For transcardial perfusion, overdose rodent species of interest with pentobarbital (120 mg/kg body weight) and transcardially perfuse them with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.76 mM KH2PO4, 10 mM Na2HPO4) followed by 4% paraformaldehyde (PFA)24.
	<ol>
		<li>Specifically, open the abdomen and the rib cage using scissors and hold the rib cage in place with Kelly hemostatic forceps to expose the heart.</li>
		<li>Insert a 23 GA needle connected to a perfusion pump into the left ventricle and quickly cut the right atrium using fine scissors.</li>
		<li>Administer PBS through the perfusion pump and the needle in the heart for 10 min to clear the brain and body of blood.</li>
		<li>Switch the perfusion pump to 4% PFA for 10 min and check for rigidity of limbs and tail to confirm successful perfusion.</li>
	</ol>
	</li>
	<li>After perfusion, decapitate the animals and remove their brain from the skull. Keep the brains overnight in 4% PFA before transferring to PBS. Embed brainstems in 4% agarose (in PBS) and slice coronally using a vibratome at 200 &#181;m thickness.</li>
</ol>
3. Staining
<ol>
	<li>Stain free floating sections for Nissl, to visualize cell bodies (1:100), in antibody media (AB media: 0.1 M phosphate buffer (PB: 50 mM KH2PO4, 150 mM Na2HPO4), 150 mM NaCl, 3 mM Triton-X, 1% bovine serum albumin (BSA)) for 30 min at room temperature on a standard laboratory shaker25.
	<ol>
		<li>Protect sections from light using aluminum foil and/or a cover. 550 nm or below wavelengths are compatible with CARS imaging (Figure 1). 
		NOTE: While we do not expect that Triton-X or other reagents have an impact on CARS imaging of lipids, additional controls with specific antibody media may be warranted.</li>
	</ol>
	</li>
	<li>PAUSE POINT: Store free floating sections (while protected from light) in PBS until imaging. Once sectioned, image brain sections within 2 weeks.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64013/64013fig01.jpg" /> 
Figure 1: CARS imaging can be combined with immunofluorescent imaging. The graphs shows that CARS imaging occurs at 660/640 nm red signal spectrum26. This wavelength is sufficiently far removed from the green, blue, or UV range, allowing for combination of the CARS signal with immunofluorescence in these ranges. Specifically, the graph also indicates the excitation and emission for Nissl tagged with blue fluorophore, which was combined with CARS during the collection of representative results for this publication. <a href="https://www.jove.com/files/ftp_upload/64013/64013fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Imaging
NOTE: The CARS laser set up contains a fiber laser that provides the 80 MHz clock, and an OPO (Optical Parametric Oscillator) laser with a tunable range of 770-990 nm with the Stokes beam fixed at 1031 nm, which are needed for collecting the CARS signal. There is one aperture for both beams.
<ol>
	<li>Before bringing samples to the microscope, turn on and warm up CARS laser for at least 1 h, align the CARS laser, and Koehler the condenser optics and the diaphragm of the microscope for forward CARS imaging. 
	NOTE: This step is critical for proper function of CARS microscopy.
	<ol>
		<li>For spatial alignment of the two laser beams (pump and stokes), access the two internal PSDs (position sensitive detectors) via the CARS laser GUI.</li>
		<li>Achieve temporal alignment by using the delay function in the CARS laser GUI, which can help overlap the pulses of the two lasers (pump and stokes) that have different dispersions because of their different wavelengths. Therefore, both the temporal and spatial overlapping of the pump and stokes beams are done with the user input through the GUI.</li>
		<li>Adjust the external periscope (last two mirrors of the setup) to center the spatially overlapped two lasers onto the scanning head mirrors of the microscope.</li>
		<li>For best forward CARS non-descanned detection, make sure the condenser is Koehler-ed (meaning the condenser is centered and focused onto the diaphragm to achieve uniform illumination)</li>
	</ol>
	</li>
	<li>For immunofluorescence confocal imaging and CARS imaging, fit the CARS laser, with both forward and epi CARS non-descanned detectors (NDDs), by incorporating a confocal microscope equipped with visible lasers for fluorescence imaging (Figure 2).</li>
	<li>Place sections in a culture dish with coverslip (for inverted microscopy), PBS to avoid the tissue drying out, and a glass weight to keep tissue near coverslip.</li>
	<li>Take z-stacks or single images with a 60X, 1.2 NA infrared corrected water objective, which serves for collection of the CARS signal in the epi direction and through a 0.55 NA condenser in the forward direction for imaging brain areas such as the medial nucleus of the trapezoid body (MNTB).
	<ol>
		<li>Take the CARS images at approximately 600 mW pump/probe and 300 mW Stokes, by using the CARS laser GUI. These laser power values are measured internally by the system. Both lasers&#39; powers at the sample location are less than 25 mW and safe for the tissue sample.</li>
		<li>Overlap the pump and Stokes beams spatially and temporally. Tune the OPO to 797.2 nm. This yields a CARS wavelength of 650 nm. Because of the higher energy level, the resulting return to the ground state is anti-Stokes (blue shifted) to the excitation.</li>
		<li>Capture the CARS signal in epi or forward non-descanned detectors using bandpass filters (640-680 nm) followed by sequential detection of the immunofluorescence label (in this instance fluorescently labeled Nissl). 
		NOTE: The Nissl neuronal soma marker is not caught in the CARS 640-680 nm bandpass filter, allowing the combination of fluorescence and CARS imaging in the images presented below.</li>
		<li>The CARS and fluorescence do not share PMTs. Use these settings for optimal lipid signal to selectively image myelination in the brain area. 
		CAUTION: Shield the user from the laser beam</li>
	</ol>
	</li>
	<li>Save the images as .oib files, which can be imported into an image analysis program for further quantification (Figure 3).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64013/64013fig02.jpg" /> 
Figure 2: CARS instrument diagram showing CARS lasers (red arrows) and non-descanned (NDD) epi and forward detection incorporated onto a laser scanning confocal. In forward NDD we acquire CARS for C-H bonds (dark red arrows) and SHG (second harmonic generatioin) at 515 nm (orange arrow). In epi NDD, we acquire CARS for C-H bonds (dark red arrows) and 2PE (two-photon emission) autofluorescence (light blue arrow). Sequentially, fluorescence confocal images can be acquired (green arrows for visible laser, blue arrows for confocal detection). <a href="https://www.jove.com/files/ftp_upload/64013/64013fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64013/64013fig03v2.jpg" /> 
Figure 3: CARS can illuminate myelin (magenta) in brain tissue (brainstem) while also imaging Nissl (cyan) or fluorescent markers. The two panels show representative results from a Mongolian gerbil (single image M. unguiculatus, Figure 3A,C,E) and mouse (z-stack max projection M. musculus, Figure 2B,D,F) brain, indicating that this technique can be used across species. Figure 3A,B showing Nissl in cyan, C,D show the CARS signal in magenta, E,F combine the Nissl and CARS signals with each panel for gerbil or mouse, respectively. Both sets of images show a section of the medial nucleus of the trapezoid body (MNTB) in the brain stem. Neurons in the MNTB receive inputs from heavily myelinated axons, which terminate in the calyx of held, a type of giant synapse27. Scale bar is 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64013/64013fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflicts of interest.

A growing body of literature emphasizes the role of myelin in brain function13,16,21,28. Moreover, we know that myelination thickness and myelination pattern can change in several neurological conditions such as multiple sclerosis (reviewed in29), aging (reviewed in30), autism20,31, and ma...

Action potentials are the basic unit of information in the brain, and action potential propagation through axons forms one pillar of information processing1,2,3. Neurons typically receive afferent inputs from multiple other neurons and integrate these inputs within a given narrow time window4,5. Therefore, the mechanisms of action potential propagation in axons have received a significant amount of attention from investigators.
When propagating through an axon, an action potential is regenerated repeatedly along the axon to ensure reliable propagation6. In most neurons of jawed vertebrates (gnathostomes) axons are surrounded by a sheath of myelin, which is a lipid-rich substance produced by nearby oligodendrocytes or Schwann cells, which are types of glial cells (reviewed in7,8). This myelin sheath electrically insulates the axon, reducing its capacitance and allowing action potential propagation efficiently, quickly, and with lower energy consumption. Myelin does not cover the axon uniformly, but it sheaths the axon in segments that have short gaps in between them, called the nodes of Ranvier (reviewed in9,10). Both myelination thickness, which controls the level of electrical insulation of an axon, and the spacing of the nodes of Ranvier, which control the frequency with which action potentials are regenerated along an axon, influence the speed of action potential propagation (reviewed in11).
There is a large body of literature suggesting that myelination thickness affects the speed of action potential propagation in axons12,13,14. Moreover, alterations in axon myelination can result in a number of CNS deficits15,16,17,18,19,20,21. It is therefore not surprising that the focus of many research efforts involves the measurement and characterization of axon myelination. Measurements of myelin thickness have most commonly been done with electron microscopy, a technique that requires a significant amount of tissue preparation and is challenging to use in combination with immunohistochemistry. However, there is also a faster and simpler technique to measure axon myelination that is based on Coherent Anti-Stokes Raman Spectroscopy (CARS). A CARS laser can be tuned to various frequencies and when tuned to frequencies that are suitable to excite lipids, myelin can be imaged without the need for any additional labels22. The lipid imaging can be combined with standard immunohistochemistry such that lipids can be imaged together with several fluorescent channels23. Imaging myelination with CARS is significantly faster than electron microscopy and has a resolution that is, albeit lower than EM, sufficient to detect even small differences in myelination in the same type of axons.

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 mL disposable syringe with needle 27 GA x 0.5&quot;</td><td>Exel int</td><td>260040</td><td></td></tr><tr><td>Fatal +</td><td>Vortech</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Surgery:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spring Scissors - 8mm Cutting Edge</td><td>Fine Science Tools</td><td>15024-10</td><td></td></tr><tr><td>Standard tweezers</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>&lt;strong&gt;Perfusion:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Fisher Chemical</td><td>SF994 (CS)</td><td></td></tr><tr><td>Fine Scissors - Sharp</td><td>Fine Science Tools</td><td>14063-11</td><td></td></tr><tr><td>Kelly hemostats</td><td>Fine Science Tools</td><td>13019-14</td><td></td></tr><tr><td>Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Needle tip, 23 GA x 1&quot;</td><td>BD precision glide</td><td>305193</td><td></td></tr><tr><td>Phosphate buffered saline (PBS):</td><td></td><td></td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Potassium phosphate monobase</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>pump with variable flow or equivalent</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Chemical</td><td>s271-1</td><td></td></tr><tr><td>Sodiumphosphate dibasic</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>&lt;strong&gt;Dissection:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mL vial with 4% PFA</td><td></td><td></td><td></td></tr><tr><td>Bochem Chemical Spoon 180mm</td><td>Bochem</td><td>230331000</td><td></td></tr><tr><td>Fine Scissors - Sharp</td><td>Fine Science Tools</td><td>14063-11</td><td></td></tr><tr><td>Noyes Spring Scissors</td><td>Fine Science Tools</td><td>15011-12</td><td></td></tr><tr><td>Pair of fine (Graefe) tweezers</td><td>Fine Science Tools</td><td>11050-10</td><td></td></tr><tr><td>Shallow glass or plastic tray, approximately 10&quot; x 10&quot;</td><td></td><td></td><td></td></tr><tr><td>Standard tweezers</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>Surgical Scissors - Blunt</td><td>Fine Science Tools</td><td>14000-20</td><td></td></tr><tr><td>&lt;strong&gt;Slicing:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agar, plant</td><td>RPI</td><td>9002-18-0</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000s</td><td></td></tr><tr><td>well plate</td><td>Alkali Sci.</td><td>TPN1048-NT</td><td></td></tr><tr><td>&lt;strong&gt;Staining:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AB Media:</td><td></td><td></td><td>1n 1,000 mL of Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Phosphate buffered (PB):</td><td></td><td></td><td></td></tr><tr><td>Potassium Phosphate Monobase</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>Sodium Phosohate Dibasic</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>BSA (Bovine serum albumin)</td><td>Sigma life science</td><td>A2153-100g</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Chemical</td><td>s271-1</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma - Aldrich</td><td>x100-500ml</td><td></td></tr><tr><td>Nissl 435/455</td><td>Invitrogen</td><td>N21479</td><td></td></tr><tr><td>&lt;strong&gt;CARS:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>APE&amp;nbsp;picoemerald laser</td><td>Angewandte Physik &amp;amp; Elektronik&amp;nbsp;GmbH</td><td></td><td></td></tr><tr><td>bandpass filter (420-520 nm)</td><td>Chroma Technology</td><td>HQ470/100m-2P</td><td></td></tr><tr><td>bandpass filter (500-530 nm)</td><td>Chroma Technology</td><td>HQ515/30m-2P</td><td></td></tr><tr><td>bandpass filters (640-680 nm)</td><td>Chroma Technology</td><td>HQ660/40m-2P</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Cut Transfer pipet</td><td>Fisher</td><td>13-711-7M</td><td></td></tr><tr><td>dichroic longpass 565 nm</td><td>Chroma Technology</td><td>565dcxr</td><td></td></tr><tr><td>dichroic longpass 585 nm</td><td>Chroma Technology</td><td>585dcxr</td><td></td></tr><tr><td>dichroic shortpass 750 nm</td><td>Chroma Technology</td><td>T750spxrxt</td><td></td></tr><tr><td>glass bottom culture dish</td><td>MatTek</td><td>P35G-0-10-C</td><td></td></tr><tr><td>glass weight (10 mm x 10 mm boro rod)</td><td>Allen Scientific Glass Inc</td><td></td><td></td></tr><tr><td>multiphoton shortpass emission filter 680 nm</td><td>Chroma Technology</td><td>ET680sp-2p8</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr></tbody></table>

coherent anti-stokes raman spectroscopy (cars) application for imaging myelination in brain slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

One of the biggest advantages of CARS microscopy over other techniques is the compatibility with fluorescent imaging23. Figure 1 shows the CARS spectra compared to Nissl tagged with immunofluorescent marker showing little/no overlap in spectra. Figure 2 illustrates the laser set up for CARS in combination with confocal microscopy. Figure 3 demonstrates two representative images, one as a single stack and one ...

Watch this Scientific Journal Video about Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices at JoVE.com

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

coherent-anti-stokes-raman-spectroscopy-cars-application-for-imaging

Research

JoVE Journal

Neuroscience

2.6K Views.  Oklahoma State University. Deze techniek kan myeline in hersenweefselsecties in beeld brengen. myelinisatie speelt een belangrijke rol bij de geleiding van actiepotentialen en het begrijpen van myeline zal ons helpen de hersenfunctie te begrijpen. Het grote voordeel van de CARS-techniek ten opzichte van bijvoorbeeld elektronenmicroscopie is dat CARS een lichtmicroscopietechniek is en gemakkelijk kan worden gecombineerd met andere fluorescentiebeelden, zoals gebruikt in de immunohistochemie.Verschillende medische...

Video: Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Engineering

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Bioengineering

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

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